We have demonstrated previously that a hairpin ribozyme targeting the 5'- leader sequence of HIV-1, when expressed stably in Jurkat cells under the control of the pol III tRNA promoter, is able to confer long-term resistance to infection by diverse strains of HIV-1, including uncloned clinical isolates (3-5). Furthermore, transduction of primary lymphocytes with the ribozyme gene delivered in a murine retrovirus vector also confers long-term resistance to infection (Leavitt et al., in preparation). In September, 1993, we obtained approval from the NIH Recombinant DNA Advisory Committee (RAC) to conduct a phase 1 trial to evaluate the safety and fate of ribozyme gene altered T-cells from HIV- infected individuals in vivo. The purpose of this SPIRAT application are two-fold: to seek support for the conduct of this phase 1 trial and to simultaneously develop preclinical studies that would lead to additional, improved clinical protocols (e.g. targeting CD34+ cells, using alternative vectors such as AAV and HIV and using combinatorial approaches as well as feedback from the clinical trial) that could be initiated within the term of the SPIRAT award. The major theme of this SPIRAT application is to develop and clinically evaluate gene therapy against HIV infection. A clinical component (D. Looney, Project 2) will initiate a Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a retrovirus vector expressing ribozyme that cleaves HIV-1 RNA and to develop clinical protocols for subsequent trials. Scientists at Immusol (M. Yu, Project 9001) will provide the retrovirus vector prepared under GMP conditions for this and subsequent trials, assist in monitoring persistence and ribozyme expression of the gene altered cells in vivo and to evaluate the antiviral effects of different combinatorial approaches in vitro. Dr. Wong-Staals group (Project 1) will focus on the development and comparisons of Adeno-associated virus vectors, HIV-vectors and a novel HIV/AAV chimeric vector for transduction efficiency and efficacy in primary lymphocytes, monocytes/macrophages and hematopoietic progenitor cells. Dr. Anthony Ho (Project 3) will focus on preclinical studies of in vitro expansion and transduction of hematopoietic stem cells with anti-HIV gene(s) and development of a Phase 1 trial for stem cell transduction.